Development of a protein-driven gene targeting technology

描述（由申请人提供）：该项目旨在通过改善基因靶向的核递送、效率和安全性并扩大其应用，改变基因和基因组在细胞内精确操纵的方式。基因靶向是一种通过将突变DNA序列改变为野生型拷贝或反之亦然而随意修饰内源DNA序列的遗传技术，而不将其从活细胞染色体中的天然背景中移除。因此，基因靶向不仅是基因、蛋白质和复杂生物系统的功能分析的基本过程，而且潜在地也是用于预防和治疗源自特定DNA改变的人类遗传疾病的分子治疗的基本过程。基因靶向的基础是遗传信息的原位交换，在目前的基因组修饰方法中，其遵循天然同源重组的步骤。不幸的是，同源重组仅在极少数生物体和细胞中是活跃和有效的，而且，它只能发生在细胞周期的有限时期。在靶向基因座处诱导DNA损伤（例如双链断裂）可以部分克服许多细胞类型（包括人细胞）中的低重组频率。然而，由于在靶向区域诱导断裂和脱靶断裂的发生，不需要的突变和重排的强烈威胁高度限制了基因治疗中的应用。事实上，双链断裂被认为是癌症的主要原因之一。本研究的目的是促进新型蛋白质-DNA复合物向细胞核的递送，以促进非典型模式下的同源配对和同源链交换反应，而不遵循同源重组的步骤，从而使基因靶向在所有可被外源DNA转化、转染或转导的细胞中有效且无损伤。我们假设，在那些通常直接或间接与染色体DNA相互作用的蛋白质中，如果与DNA靶向分子结合，可以促进该分子的递送和靶向效率。我们开发和测试这一假设的具体目的是：目的1）测试DNA靶向分子的不同设计，并确定最有效的一种。目的2）制备含有位点特异性DNA结合蛋白的DNA-蛋白质复合物，以驱动DNA模板分子到达靶位点。我们将使DNA靶向分子与已知在所选靶向位点附近以序列特异性方式结合DNA的蛋白质复合。目标3）识别一组蛋白质，当与DNA模板分子结合时，可以[模块化]促进染色体基因靶向。这将通过A）使用由酵母细胞提取物产生的cDNA文库在酵母细胞中筛选基因靶向促进蛋白，和通过B）使用由人癌症干细胞的细胞提取物产生的cDNA文库在对损伤修复敏感的人细胞中直接筛选基因靶向促进蛋白来实现。[C)测试鉴定的GTP促进基因靶向的模块化能力。D）在蛋白质驱动的基因靶向之后测试细胞的基因组稳定性。 公共卫生相关性：与特定遗传缺陷相关的疾病的最佳治疗/预防以及研究基因功能的最佳方法可以通过基因靶向对所需基因进行精确的原位修饰来实现。然而，基因靶向通常是一个效率低下的过程，特别是在人类细胞中。已知DNA双链断裂（DSB）有效地刺激同源靶向。然而，DSB诱导酶的切割特异性是一个主要问题，因为即使是很少的脱靶DSB也易于产生突变和染色体重排，这可能导致癌症。我们的目标是开发一种模块化的、无癌症的和有效的基因靶向方法，其中DNA靶向分子通过促进核递送并促进链交换反应而不诱导DNA损伤的蛋白质直接驱动到其基因组靶标。这是一项探索性研究，所获得的结果将为未来R 01申报提供基础，该申报重点关注蛋白质引导的基因靶向机制以及基因和基因组修饰和基因治疗的更广泛应用。

DESCRIPTION (provided by applicant): This project aims to transform the way in which genes and genomes can be precisely manipulated inside cells by improving the nuclear delivery, efficiency and safety of gene targeting and expanding its applications. Gene targeting is a genetic technique to modify endogenous DNA sequence at will, by changing a mutant DNA sequence into a wild-type copy or vice versa, without removing it from its natural context in the chromosome in a living cell. Gene targeting is therefore a fundamental process not only for functional analysis of genes, proteins and complex biological systems, but potentially also in molecular therapy for the prevention and cure of human genetic diseases originating from specific DNA alterations. The basis of gene targeting is the in situ exchange of genetic information, which in current approaches of genome modification follows the steps of natural homologous recombination. Unfortunately, homologous recombination is active and efficient only in an extremely small set of organisms and cells, where, moreover, it can occur only in a restricted period of the cell cycle. The induction of DNA damage such as double-strand breaks at the targeting locus can partially overcome the low recombination frequencies in many cell types, including human cells. However, the strong threat of unwanted mutations and rearrangements due to both the induction of a break at the targeting region and the occurrence of off-target breaks highly limits applications in gene therapy. In fact double-strand breaks are regarded as one of the primary causes of cancer. The goal of this study is to promote the delivery of novel protein-DNA complexes to the nucleus to promote the reactions of homologous pairing and homologous strand exchange in a non canonical mode without following the steps of homologous recombination, thereby making gene targeting efficient and damage-free in all cells that can be transformed, transfected, or transduced by exogenous DNA. We hypothesize that several proteins among those that normally directly or indirectly interact with chromosomal DNA, if bound to a DNA targeting molecule, can promote delivery and targeting efficiency of this molecule. Our Specific Aims to develop and test this hypothesis are: Aim 1) Test different designs for the DNA targeting molecules and identify the most effective one. Aim 2) Generate DNA-protein complexes containing a site-specific DNA binding protein to drive the DNA template molecule to the targeting locus. We will complex DNA targeting molecules with proteins that are known to bind the DNA in a sequence specific manner in the vicinity of a chosen targeting locus. Aim 3) Identify a set of proteins that, when bound to a DNA template molecule, can [modularly] boost chromosomal gene targeting. This will be achieved via A) screening for gene targeting promoting proteins in yeast cells using a cDNA library generated from yeast cell extract, and via B) screening for gene targeting promoting proteins directly in human cells sensitized to damage repair using a cDNA library generated from cell extract of human cancer stem cells. [C) Testing the identified GTPs for their modular capacity to promote gene targeting.] D) Testing the genomic stability of cells following protein- driven gene targeting. PUBLIC HEALTH RELEVANCE: The best cure/prevention of diseases associated with specific genetic defects as well as the best approach to study gene function can be achieved by precise in situ modification of the desired gene/s, via gene targeting. However, gene targeting is mostly an inefficient process, especially in human cells. DNA double-strand breaks (DSBs) are known to efficiently stimulate homologous targeting. However, cleavage specificity of DSB- inducing enzymes is a major problem since even few off-target DSBs are prone to generate mutations and chromosome rearrangements, which can lead to cancer. Our goal is to develop a modular, cancer-free and efficient gene targeting approach where the DNA targeting molecule is directly driven to its genomic target by a protein that facilitates nuclear delivery and promotes the reaction of strand exchange without inducing DNA damage. This is an exploratory study and the results obtained will provide a basis for a future R01 submission focused on mechanisms of gene targeting guided by proteins and on broader application for gene and genome modification and gene therapy.